Evidence for two genetically distinct DNA primase activities specified by plasmids of the B and I incompatibility groups.

Soluble formate dehydrogenase from Methanobacterium formicicum was purified 71-fold with a yield of 35%. Purification was performed anaerobically in the presence of 10 mM sodium azide which stabilized the enzyme. The purified enzyme reduced, with formate, 50 mumol of methyl viologen per min per mg of protein and 8.2 mumol of coenzyme F420 per min per mg of protein. The apparent Km for 7,8-didemethyl-8-hydroxy-5-deazariboflavin, a hydrolytic derivative of coenzyme F420, was 10-fold greater (63 microM) than for coenzyme F420 (6 microM). The purified enzyme also reduced flavin mononucleotide (Km = 13 microM) and flavin adenine dinucleotide (Km = 25 microM) with formate, but did not reduce NAD+ or NADP+. The reduction of NADP+ with formate required formate dehydrogenase, coenzyme F420, and coenzyme F420:NADP+ oxidoreductase. The formate dehydrogenase had an optimal pH of 7.9 when assayed with the physiological electron acceptor coenzyme F420. The optimal reaction rate occurred at 55 degrees C. The molecular weight was 288,000 as determined by gel filtration. The purified formate dehydrogenase was strongly inhibited by cyanide (Ki = 6 microM), azide (Ki = 39 microM), alpha,alpha-dipyridyl, and 1,10-phenanthroline. Denaturation of the purified formate dehydrogenase with sodium dodecyl sulfate under aerobic conditions revealed a fluorescent compound. Maximal excitation occurred at 385 nm, with minor peaks at 277 and 302 nm. Maximal fluorescence emission occurred at 455 nm.     

Demonstration of a third incompatibility function on plasmids already incompatible with group P and group I plasmids

The addition of extra antibiotic resistance determinants to plasmids of a series previously shown to be incompatible in an atypical fashion with group I and group P plasmids, has enabled the existence of a third and major incompatibility function determined by these plasmids to be demonstrated. The in vitro construction of a plasmid consisting of the replication region of one of the plasmids, linked to the genes of the galactose operon, facilitated the identification of this third incompatibility function as similar to IncB plasmids.     

Isolation of large bacterial plasmids and characterization of the P2 incompatibility group plasmids pMG1 and pMG5.

Large plasmids from Agrobacterium tumefaciens, Salmonella typhimurium, Escherichia coli, Pseudomonas putida, and Pseudomonas aeruginosa were routinely and consistently isolated using a procedure which does not require ultracentrifugation but includes steps designed to separate large-plasmid DNA from the bacterial folded chromosome. It also selectively removes fragments of broken chromosome. A variety of large plasmids was readily visualized with agarose gel electorphoresis, including five between 70 and 85 megadaltons (Mdal) in size, six between 90 and 143 Mdal, one that was larger than 200 Mdal, and one that was larger than 300 Mdal. This isolation procedure allowed initial estimation of the molecular sizes of the two IncP2 plasmids, pMG1 and pMG5, which were 312 and 280 Mdal, respectively. A standard curve for size determination by gel electrophoresis including plasmids between 23 and 143 Mdal in size did not extrapolate linearly for plasmids of the 300-Mdal size range. Unique response of different plasmids to the isolation procedure included sensitivity of IncP1 plasmids to high pH and the co-isolation of a 20-Mdal "cryptic" plasmid in conjunction.     

Serological characteristics of pili determined by the plasmids R711b and F0lac.

The plasmids R711b (at present IncX) and F0lac (IncFV) both determine pili morphologically like those of F (IncFI), and confer sensitivity to the F-specific filamentous bacteriophages, but not to the F-specific isometric RNA phages. Detailed serological studies show that the two pilus types are unrelated, and that neither is related to any of the previously defined F pilus serotypes. Adsorption of the isometric RNA phage MS2 to R711b pili occurs in the presence but not in the absence of formalin, which presumably prevents elution of reversibly adsorbed virions. No adsorption occurs with F0lac pili. MS2 multiplication, as measured by titre increase tests in liquid medium, is found with neither plasmid. The two plasmids are not incompatible. These observations indicate that R744b and F0lac are different both from one another and from the plasmids belonging to the incompatibility groups IncFI--IV.     

Characterization of pili determined by drug resistance plasmids R711b and R778b.

The bacterial drug resistance plasmids R711b and R778b, at present classified in the X incompatibility group, determine pili (designated 711) that resemble F pili morphologically. Like F pili, 711 pili adsorb F-specific filamentous bacteriophages to their tips, though more often in pairs, than singly. However, F-specific RNA-containing bacteriophages are not adsorbed to their sides, and strains carrying the plasmids are resistant to these phages. Pili determined by the only IncFV plasmid Folac are similar to 711 pili in their phage adsorption properties, but they are serologically different, as are F pili. It is concluded that F, Folac and 711 pili have basic differences in spite of a morphological resemblance.     

Morphology of pili determined by the N incompatibility group plasmid N3 and interaction with bacteriophages PR4 and IKe

The IncN plasmid N3 was transferred to bald strains of Salmonella typhimurium LT2 and Escherichia coli K-12. In both cases, transconjugants were found to carry short pili, which were designated N pili. They were very easily detached from cells and readily broken into short pieces. N pili were found to be straight inflexible rods 9.5 nm thick and sharply pointed at the distal end. The N-specific filamentous bacteriophage IKe and the lipid-containing phage PR4 both adsorbed to the pointed tips when mixed with cell-free suspensions of N pili.     

Suppression and enhancement of temperature sensitivity of dnaB mutations of Escherichia coli K12 by conjugative plasmids.

A majority of wild-type, conjugative antibiotic-resistance plasmids from a standard collection had the ability to suppress or to enhance the temperature sensitivity of dnaB mutants of Escherichia coli K12. This ability appears to be widely dispersed among all plasmid groups. The mode of suppression does not involve the insertion of the plasmid into the chromosome. Plasmid-induced suppression or enhancement is not as a rule mutation specific and can extend to several mutations within the dnaB region. The patterns of suppression and enhancement suggest a direct or indirect interaction between a plasmid-specified product and the dnaB protein.     

Restriction endonuclease analyses of the incompatibility group P-1 plasmids RK2, RP1, RP4, R68, and R68.45

The P-group plasmids RP1, RP4, RK2, R68 and R68.45 were analyzed by the following restriction endonucleases:BamHI,BglII,EcoRI,HindIII,PstI,PvuII,SalI, andSmaI. No differences between RP1, RP4, and RK2 were found, and the plasmid R68.45 was found to contain a direct duplication of an existing DNA sequence in R68. Our map of RK2 differs from the published map of RK2 in the corresponding region of the R68 map that is duplicated in R68.45.     

Evidence that the hemolysin/bacteriocin phenotype of Enterococcus faecalis subsp. zymogenes can be determined by plasmids in different incompatibility groups as well as by the chromosome.

The hemolysin (Hly/Bac) determinant in strains of Enterococcus faecalis was found to be present on plasmids in different incompatibility groups (conferring different sex pheromone responses) as well as on the chromosome. Of 33 Hly/Bac plasmids identified in clinical isolates, the related pheromone for 30 was cAD1; the related pheromone for another two (pYI1 and pYI3) or one (pYI2) was cOB1 or cY12, respectively. The representative Hly/Bac plasmids pAD1, pYI1, pOB1, and pYI2, which responded to pheromones cAD1, cOB1, cOB1, and cYI2, respectively, were compatible with one another. As additions to the incompatibility group IncHly of pAD1, groups for pOB1, pYI1, and pYI2 were designated IncHlyII, IncHlyIII, and IncHlyIV, respectively. Eleven of the 30 plasmids conferring a response to cAD1 were very similar to pAD1 on the basis of their restriction endonuclease profiles. EcoRI fragment D, F, or H containing parts of the Hly/Bac gene(s) of pAD1 hybridized to similar EcoRI fragments from each of the other three representatives of incompatibility groups (i.e., pOB1, pYI1, and pYI2) and to homologous DNA representing the chromosome of the plasmid-free Hly/Bac strain YI6-1.     

Generation of the HLA-B35, -B5, -B16, and B15 groups of alleles studied by intron 1 and 2 sequence analysis

 HLA-B is the most polymorphic of the major histocompatibility complex classical class I loci. This polymorphism is mainly in exons 2 and 3, which code for the molecule’s α1 and α2 domains and include the antigenic peptide binding site. Recent studies have indicated that not only exons but also the intron 2 region may be involved in the generation of certain HLA-B alleles such as B ^* 3906 and B ^* 1522. To study the degree of intron 2 participation and the mechanisms that generate polymorphism at the HLA-B locus, intron 1 and 2 sequences from the HLA-B35, -B5, -B16 and -B15 groups of alleles were obtained. A group-specific intronic polymorphism was found: namely, B ^* 5301 shows intron 1 and 2 sequences identical to those found in all B35 alleles studied. On the other hand, B ^* 5101 and B ^* 52012 show the same intron 1 and 2 sequences and their intron 1 is the same as that found in the B35 group. This suggests that B5 and B35 groups of alleles may have arisen from a common ancestor. All known B16 alleles show the same introns 1 and 2, with the exception of B ^* 39061 and B ^* 39062, and all B15 alleles also bear the same introns 1 and 2, with the exception of B ^* 1522. Variability at intron 1 is more restricted than at intron 2, and the use of intron 1 for HLA-B allele phylogenetic analysis is better for grouping alleles of a postulated common origin. In conclusion, there is a remarkable conservation of intronic sequences within related HLA-B alleles, which probably reflects a common origin and perhaps a selective force avoiding DNA changes. Intronic sequences are also potentially useful to design DNA typing strategies. Received: 11 March 1997 / Revised: 29 May 1997     

Cytochalasins Z1, Z2 and Z3, three 24-oxa[14]cytochalasans produced by Pyrenophora semeniperda

Three new cytochalasans, named cytochalasins Z1, Z2 and Z3, were isolated from the wheat culture of Pyrenophora semeniperda, a fungus proposed to biologically control grass weeds. Other cytochalasins isolated from the same organic extract were identified as the already known cytochalasins F, T, deoxaphomin and cytochalasins B, the latter being produced in very large amounts. All three new cytochalasins were characterized as 24-oxa[14]cytochalasans by extensive use of NMR and MS techniques. Cytochalasins Z1 and Z2 proved to be structurally related to cytochalasin T, whereas cytochalasin Z3 was related to cytochalasin B. When assayed on wheat and tomato seedlings, cytochalasin Z3, in comparison to the new cytochalasins, cytochalasin B, its 21,22-dihydroderivative, cytochalasin F and deoxaphomin showed a remarkable ability to inhibit root elongation. The possibility of using these metabolites in biological control strategies is discussed.     

Expression of unilateral incompatibility in pollen of Lycopersicon pennellii is determined by major loci on chromosomes 1, 6 and 10

Summary We have previously described gene introgression from the wild nightshade Solanum lycopersicoides into tomato (Lycopersicon esculentum) through the use of either diploid or sesquidiploid hybrids (the latter consisting of two genomes of L. esculentum and one genome of S. lycopersicoides). Both types of intergeneric hybrids display pollen sterility, but workable ovule fertility. Unilateral incompatibility prevents their direct hybridization with staminate L. esculentum. Pollen of a self-compattible form of the related wild species L. pennellii is compatible with pistils of L. esculentum x S. lycopersicoides hybrids. This trait was backcrossed from L. pennellii to L. esculentum in order to develop bridging lines that could be used to obtain progeny from the intergeneric hybrids and to study the inheritance of bridging ability. In progeny of L. esculentum x S. lycopersicoides hybrids pollinated with L. pennellii-derived bridging lines, preferential transmission of L. pennellii alleles was observed for certain isozyme and RFLP markers on chromosomes 1, 6 and 10. The skewed segregations suggest linkage to three major pollen-expressed compatibility loci. This was confirmed by observations of pollen tube growth, which indicated that compatibility with pistils of the diploid intergeneric hybrid occurred only in bridging lines at least heterozygous for the L. pennellii markers on chromosomes 1, 6 and 10. Compatibility with the sesquidiploid hybrid required only the chromosome 1 and 6 loci, indicating an apparent effect of gene dosage on expression of incompatibility in the pistil. In an F₂ L. esculentum x L. pennellii population, preferential transmission of L. pennellii alleles was observed for the same markers on chromosomes 1 and 10, as well as other markers on chromosomes 3, 11, and 12, but not 6. The chromosome 1 pollen compatibility locus maps to or near the S-locus, which determines S-allele specificity. The results are discussed in relation to existing genetic models for unilateral incompatibility, including the possible involvement of the S-locus.     

Regioselective synthesis of 1I,1II,5I,5II,6I,6I,6II,6II-2H8-cellobiose

Partially deuteriated 1,5,6,6-(2)H(4)-d-glucose and 1(I),1(II),5(I),5(II),6(I),6(I),6(II),6(II)-(2)H(8)-d-cellobiose were synthesized in high yields and on a large scale from d-glucose. (2)H enrichment at C-5 and C-6 of each glucopyranosyl unit in excess of 85% and 90%, respectively, was realized by (1)H-(2)H exchange in (2)H(2)O containing deuteriated Raney Ni. Nucleophilic addition of LiAlD(4) to 5,6,6-(2)H(3)-2,3,4,6-tetra-O-benzyl-d-gluconolactone led to a 98% (2)H enrichment at C-1. Deuteriated cellobiose is of interest as building block for the synthesis of a model compound of cellulose I.     

Recognition of B and Z forms of DNA by Escherichia coli DNA polymerase I

Since the substrate binding domain of the large proteolytic fragment of Escherichia coli DNA polymerase I has been shown to interact with the B forms of DNA, we have studied the ability of this enzyme to recognize structures other than the B form. The polymerase activity has been used to evaluate the degree of recognition of the B and Z forms of DNA. The Z form was found to promote less activity, indicating the probable inability of the polymerase to move along the conformationally rigid form of the template. The present study indicates that the Z-DNA found in vivo may have a role in the control of replication.     

Immune Checkpoints of the B7 Family. Part 1. General Characteristics and First Representatives: B7-1, B7-2, B7-H1, B7-H2, and B7-DC

Russian Journal of Bioorganic Chemistry - T-cell response along with humoral response compose the basis of acquired immunity. Effective activation of T lymphocytes requires at least two signals....